Method for treating the ear, nose, sinus or throat

ABSTRACT

Bodily tissue and structures may be protected using a fluid layer containing a mixture of chitosan and oxidized polysaccharide. The mixture forms a protective gel layer via in situ crosslinking. Compared to crosslinking using a low molecular weight aldehyde such as glutaraldehyde or genipin, oxidized polysaccharides appear to provide faster gelation while avoiding the use of potentially less bioacceptable low molecular weight aldehydes.

CROSS REFERENCE TO RELATED APPLICATION

This application is a Divisional of U.S. patent application Ser. No.12/429,141 filed Apr. 23, 2009 which claims priority from U.S.Provisional Application Ser. No. 61/047,590 filed Apr. 24, 2008, thedisclosures of which are incorporated herein by reference.

FIELD OF THE INVENTION

This invention relates to polysaccharides and to materials for use in oron tissue and structures in the ears, nose, throat, limbs and spinalcolumn.

BACKGROUND

Certain polysaccharide materials have been used for surgical repair ordrug delivery. Documents relating to such materials include U.S. Pat.No. 6,514,522 (Domb) and U.S. Pat. No. 7,053,068 B2 (Prinz), U.S. PatentApplication Publication Nos. US 2005/0176620 A1 (Prestwych et al.) andUS 2005/0238702 A1 (Ishihara et al.), Canadian Patent Application No. 2348 842 A1 (Bernkop-Schnürch), Published PCT Application Nos. WO98/31712 A2 (B.F. Goodrich Co.), WO 01/00246 A2 (Bentley et al.) and WO03/020771 A1 (Mucobiomer Biotechnologische Forschungs-und EntwicklungsGmbH), Mi et al., Synthesis and Characterization of a NovelChitosan-Based Network Prepared Using Naturally-Occurring Crosslinker, JPolym Sci, Part A: Polym Chem, 38, 2804-2814 (2000), Mi et al.,Synthesis and characterization of biodegradable TPP/genipinco-crosslinked chitosan gel beads, Polymer, 44, 6521-30 (2003), Roldo etal., Mucoadhesive thiolated chitosans as platforms for oral controlleddrug delivery: synthesis and in vitro evaluation, European Journal ofPharmaceutics and Biopharmaceutics, 57, 115-121 (2004), Krauland et al.,Viscoelastic Properties of a New in situ Gelling Thiolated ChitosanConjugate, Drug Development And Industrial Pharmacy, 31, 885-893 (2005),Bernkop-Schniirch, Thiomers: A new generation of mucoadhesive polymers,Advanced Drug Delivery Reviews, 57, 1569-1582 (2005), Bernkop-Schnürchet al., Thiomers: Preparation and in vitro evaluation of a mucoadhesivenanoparticulate drug delivery system, International journal ofPharmaceutics, 317, 76-81 (2006) and Weng et al., RheologicalCharacterization of in Situ Crosslinkable Hydrogels Formulated fromOxidized Dextran and N-Carboxyethyl Chitosan, Biomacromolecules, 8,1109-1115 (2007).

SUMMARY OF THE INVENTION

The present invention provides, in one aspect, a fluid layer atop abodily tissue or structure, the layer comprising chitosan and oxidizedpolysaccharide in amounts sufficient to form a protective gel layer insitu. The protective gel layer may assist in returning an injured,inflamed or surgically repaired tissue surface to a normal state, e.g.,through one or more healing mechanisms such as modulation of aninflammatory response, phagocytosis, mucosal remodeling, reciliation orother full or partial restoration of normal function.

The present invention provides in another aspect a method for treating abodily tissue or structure, which method comprises:

-   -   a) applying to such tissue a fluid layer containing a mixture of        chitosan and an oxidized polysaccharide, and    -   b) allowing the mixture to form a protective gel layer in situ.

The disclosed fluid layer desirably is spray applied, and packaged in amulticomponent spray dispenser. The disclosed method and layer areespecially useful for treating mucosal tissues in the ears, nose orthroat and openings, recesses, passageways or joints in the limbs orspinal column.

BRIEF DESCRIPTION OF THE DRAWING

FIG. 1 is a schematic view showing the disclosed method;

FIG. 2 is a perspective view of a dispensing instrument which may beused in the disclosed method;

FIG. 3 is a graph showing the antimicrobial properties of two in situcrosslinked gel layers formed from chitosan and oxidized polysaccharide,and of a trypticase soy broth control;

FIG. 4 is a graph showing antimicrobial activity as a function of timefor three in situ crosslinked gel layers formed from chitosan andoxidized polysaccharide, and for a trypticase soy broth control;

FIG. 5 is a graph showing drug release behavior for three in situcrosslinked gel layers; and

FIG. 6 is a graph showing degradation of an in situ crosslinked gellayer.

Like reference symbols in the various figures of the drawing indicatelike elements. The elements in the drawing are not to scale.

DETAILED DESCRIPTION

The following detailed description describes certain embodiments and isnot to be taken in a limiting sense. All weights, amounts and ratiosherein are by weight, unless otherwise specifically noted. The termsshown below have the following meanings:

The term “adhesion” refers to the sticking together of a body structureor prosthetic material to tissue, to the sticking together of tissue totissue with which it is in intimate contact for extended periods, or tothe formation of tissue that connects body structures, prostheticmaterials or tissues to one another across a normally open space.

The term “antimicrobial” refers to an ability to cause greater than a90% numeric reduction (viz., at least a 1-log order reduction) in apopulation of one or more of Staphylococcus aureus, Pseudomonasaeruginosa, Streptococcus pneumonia, Haemophilus influenzae or Moraxellacatarrhalis.

The terms “attached” and “adhered” when used in reference to a bacterialbiofilm and a surface mean that the biofilm is established on and atleast partially coats or covers the surface, and has some resistance toremoval from the surface. As the nature of this relationship is complexand poorly understood, no particular mechanism of attachment oradherence is intended by such usage.

The term “bacterial biofilm” means a community of bacteria attached to asurface, with the organisms in the community being contained within anextracellular polysaccharide (EPS) matrix produced by the bacteria.

The term “biocompatible” when used in reference to a substance meansthat the substance presents no significant deleterious or untowardeffects upon the body.

The term “biodegradable” when used in reference to a substance meansthat the substance will degrade or erode in vivo to form smallerchemical or physical species. Such degradation process may be enzymatic,chemical or physical.

The term “bioresorbable” when used in reference to a substance meansthat the substance is capable of being absorbed by the body.

The term “cohesive” when used in reference to a liquid or gel means thatthe liquid or gel when placed on a level surface will tend to (but neednot in all cases) stick to itself and form a unitary mass.

The term “comminuted” when used in reference to a particulate materialmeans that the particles have been fractured and reduced in size bycutting, grinding, pulverizing, triturating or other particle fracturingprocess employing externally-applied force.

The term “conformal” when used in reference to a composition applied totissue or other body structure means that the composition can form asubstantially continuous layer over an area to which the composition hasbeen applied.

The terms “detaching”, “removing” and “disrupting” when used inreference to a bacterial biofilm attached or adhered to a surface meanthat at least a significant amount of the biofilm initially present onthe surface no longer is attached or adhered to the surface. Noparticular mechanism of detachment, removal or disruption is intended bysuch usage.

The term “fluid” when used in reference to a substance means that thesubstance is a liquid having a loss modulus (G″) greater than itsstorage modulus (G′) and a loss tangent (tan δ) greater than 1.

The term “gel” when used in reference to a substance means that thesubstance is deformable (viz., is not a solid), G″ is less than G′ andtan δ is less than 1.

The term “gelation” when used with respect to formation of a gel layermeans the time at which G″ equals G′ and tan δ equals 1.

The term “hemostat” means a device or material which stops blood flow.

The term “hydrogel” when used in reference to a gel means that the gelis hydrophilic and contains water.

The term “hydrated” when used in reference to a device or substancemeans that the device or substance contains uniformly distributedchemically-bound water. A “fully hydrated” device or substance isincapable of taking up additional water of hydration. A “partiallyhydrated” device or substance is capable of taking up additional waterof hydration.

The term “inner ear” means the semicircular canals and cochlea.

The term “middle ear” means the region defined by the tympanic membrane,interior structures such as the ossicular chain, the surrounding liningand bordering structures such as the mastoid.

The term “mucoadhesive” when used in reference to a device or substancemeans that the device or substance will adhere to the mucus coveringepithelia.

The term “nasal or sinus cavities” refers to the various tissuesdefusing the normally air-filled passages and chambers within the noseand sinus including but not limited to the nostrils or nares, the nasalconcha or turbinates, the frontal, ethmoid, sphenoid and maxillarysinuses, the sinus ostia and the nasopharnyx.

The term “polysaccharide” includes derivatives of polysaccharides andmodified polysaccharides, as well as derivatives of individualpolysaccharide species and modified individual polysaccharide species.For example, the term “carboxymethylcellulose” includescarboxymethylcellulose derivatives and modified carboxymethylcelluloses,the term “chitosan” includes chitosan derivatives and modifiedchitosans, and the term “starch” includes starch derivatives andmodified starches.

The term “protective” when used in reference to a layer of a compositionatop tissue or other body structure means that the layer may assist inreturning an injured, inflamed or surgically repaired tissue surface toa normal state, e.g., through one or more healing mechanisms such asmodulation of an inflammatory response, phagocytosis, mucosalremodeling, reciliation or other full or partial restoration of normalfunction.

The term “residence time” when used in reference to a protective gellayer atop tissue or other body structure means the time period duringwhich the gel layer or portion thereof remains in place in vivo undergross observation.

The term “solvating” means to form a solution or dispersion containing asolvent or other carrier within which a solute is dissolved orsuspended.

The term “substantially collagen-free” means containing a sufficientlylow amount of collagen so as not to pose a potential risk oftransmission of or infection with bovine spongiform encephalopathy (BSE)or variant Creutzfeldt-Jakob disease (vCJD).

The term “thin” when used in reference to a protective layer atop tissueor other body structure means having an average thickness less thanabout two millimeters.

Referring to FIG. 1, the disclosed method may be performed for examplein the nasal or sinus cavities 100 of a patient, including the maxillarysinuses 110 a, 110 b and frontal sinuses 112 a, 112 b, which may beaccessed through nares 114 a, 114 b. It should be noted that externalfeatures of the patient, including nares 114 a, 114 b, are shown indashed lines. When the patient suffers for example from chronicrhinosinusitis, one or more treatment sites such as treatment site 116associated with a surface of maxillary sinus 110 a may be medically orif need be surgically addressed. Treatment site 116 includes ciliatedepithelium of maxillary sinus 110 a and may include an associated layerof bacteria inhabiting an associated biofilm (not shown in FIG. 1). Thetreatment site need not be natural tissue and may instead be anartificial structure (not shown in FIG. 1) such as a sinus packing orstent which may also be covered at least in part with a layer ofbacterial biofilm. If present, the biofilm may be removed using asolvating system (for example, the solvating system described in U.S.Patent Application Publication No. US 2007/0264310 A1) which may beapplied to treatment site 116 using an introducer 120 with anarticulatable delivery tube 122 containing an irrigation duct (hidden inFIG. 1) through which the solvating system may flow to a nozzle 124 atthe distal end of introducer 122 and thence to the treatment site. Thesolvating system and residues of the biofilm may be removed from thetreatment site via an aspiration duct (hidden in FIG. 1). The disclosedcomposition comprising chitosan and oxidized polysaccharide may likewisebe applied at the treatment site using the same or a differentirrigation duct in introducer 120. Those skilled in the art willappreciate that the disclosed composition (and if used, the solvatingsystem) may be applied to the treatment site using other methods ordevices. Exemplary other methods include power spray or other sprayapplication, lavage, misting, mopping, wicking, dripping andtrephination and exemplary other devices include spray nozzles (e.g.,single component or multiple component spraying nozzles) and syringes(e.g., single barrel or multiple barrel glass or plastic syringes andbulb syringes). The treatment method may also be performed in otherparts of the body. The treatment method has particular utility innon-vascular applications, including treatment of tissues (e.g., mucosaltissues) or structures in or near the ears, throat, limbs or spinalcolumn.

FIG. 2 shows an exemplary instrument 200 which may be used in thedisclosed treatment method. Instrument 200 includes a handle 202 and anintroducer 222 whose distal end 224 (referenced generally) includes aspray nozzle, irrigation and aspiration ducts (not separately numberedin FIG. 2). Instrument 200 can optionally further include a firstactuator assembly 226 (referenced generally) and a second actuatorassembly 228 (referenced generally). A control wheel 230 in firstactuator assembly 226 may be operable by a user to effectuate bending ofthe introducer 222, and a control wheel 232 in second actuator assembly228 may be operable by a user to effectuate movement or rotationrelative to introducer 222 of liquid sprayed from distal end 224 ofintroducer 222. Handle 202 serves generally as a housing for variousother components of instrument 200 and as a support for introducer 222.Handle 202 may have a pistol grip-like shape, defining a grip portion234 and a nose 236. Grip portion 234 is sized and shaped for grasping bya user's hand, whereas nose 236 is adapted for connection to introducer222. Trigger 238 and an associated sensor and valve (not shown in FIG.2) may be used to control the flow of the disclosed rehydrated gel (andif used, the disclosed solvating system) through irrigation tubing 240and thence through the spray nozzle in distal end 224 and onto thedesired treatment site. Trigger 238 may be provided with amultidirectional range of motion and associated with one or moreadditional sensors and valves (not shown in FIG. 2) to control removalfrom a treatment site of the solvating system, biofilm residue and otherdebris through the aspiration duct in distal end 224 and thence toaspiration tubing 242. Trigger 238 may also be used to control the flowof the disclosed rehydrated gel through a separate lumen in irrigationtubing 240 and thence through the spray nozzle in distal end 224 andonto the desired treatment site.

The applied composition comprising chitosan and oxidized polysaccharidemay fill the treatment site (e.g., a nasal or sinus cavity, or anopening, recess, passageway or joint in a portion of the limbs or spinalcolumn), in which case the disclosed layer of such composition may bevery thick and not exposed to air or other nearby gases, and withdiffering thicknesses throughout the layer. The disclosed compositionmay also be applied as a thin film or other conformal coating in whichcase the disclosed layer may be relatively thin and exposed to air orother nearby gases, and with a substantially uniform thicknessthroughout the layer. After gelation the protective gel layer may beviscous, elastic or viscoelastic. The protective gel layer desirablyadheres to mucosal or other natural tissues (e.g., cartilage or bone) atthe treatment site and resists detachment or other disruption untilnatural degradation or resorption of the gel layer takes place, e.g.,after a residence time in vivo of from one day to a few (e.g., 2, 3 or4) days, weeks or months. Meanwhile bacterial recolonization orreinfection may be significantly reduced or prevented, and improvedhealing and reciliation may take place. The protective gel layer mayprovide various therapeutic advantages including but not limited tobacterial adhesion repellence, anti-infective properties, local immunemodulation, tissue protection, reduction or elimination of pain orbleeding, reduction in inflammation, optimization of environment forciliary regrowth, reduction in adhesions to critical anatomy, and thelike. These advantages may arise due to a variety of mechanismsincluding a) killing bacteria, b) inhibiting bacterial colonization, c)inhibiting the adherence of bacteria to tissue, d) reducing tissuemorbidity or abscess formation, e) reducing or preventing diseaserecurrence (for example, specifically reducing the chronic inflammationrelated to bacterial toxin and EPS), f) coating and protecting tissueduring healing, such as by maintenance of a moist wound which promotesplatelet aggregation, or by closure of a dry wound without excessivescabrous formation, g) hemostasis, h) optimizing the environment forreciliation of the mucosa, i) speeding the growth or regrowth of ciliaand j) delivering therapeutic agent(s) to the treatment site. Desirablythe protective gel layer will adhere to a portion of the mucosa whileleaving the cilia in unadhered portions free to undergo natural rhythmiccilia motion (viz., cilia beating), will if desired also deliverantimicrobial agents or additional therapeutic agents, and desirablywill discourage or prevent bacteria from adhering to the treatment site.

A wide variety of chitosans (including salts and other chitosanderivatives) may be employed in the disclosed fluid layer and method.Exemplary unmodified chitosans and their salts (including citrate,nitrate, lactate, phosphate, chloride and glutamate salts) may beobtained from a variety of commercial sources including KitoZyme S.A.,Fluka Chemie AG, the NovaMatrix unit of FMC BioPolymer AS andSigma-Aldrich Co. Chitosan may also be synthesized by deacetylation ofchitin (poly-N-acetyl-D-glucosamine) to eliminate acetyl groups on thenitrogen atom by hydrolysis. The resulting polymer has a plurality ofrepeating units (e.g., about 30 to about 3000 repeating units, about 60to about 600 repeating units, or such other amount as may be desired forthe chosen end use) some or all of which contain deacetylated aminogroups (e.g., about 30 to about 100% or about 60 to about 95% of thetotal repeating units), with the remaining repeating units (if any)containing acetylated amino groups. The polymer is cationic and may beregarded as being composed from glucosamine monomers. The chitosan mayhave a variety of number average molecular weights, e.g., about 5 toabout 2000 kDa, about 10 to about 500 kDa, or about 10 to about 100 kDa.The chitosan may for example be an ultralow molecular weight materialhaving a number average molecular weight less than about 50 kDa, a lowmolecular weight material having a number average molecular weight ofabout 50 to about 200 kDa, a medium molecular weight material having anumber average molecular weight of about 200 to about 500 kDa or a highmolecular weight material having a number average molecular weightgreater than about 500 kDa. Chitosan derivatives may also be employed,for example derivatives in which one or more hydroxyl or amino groupshave been modified for the purpose of altering the solubility ormucoadhesion characteristics of the derivative. Exemplary derivativesinclude thiolated chitosans, and non-thiolated chitosan derivatives suchas acetylated, alkylated or sulfonated chitosans (for example O-alkylethers, O-acyl esters, cationized trimethyl chitosans and chitosansmodified with polyethylene glycol). Chitosan derivatives may be obtainedfrom a variety of sources. For example, thiolated chitosans may beobtained from ThioMatrix Forschungs Beratungs GmbH and MucobiomerBiotechnologische Forschungs-und Entwicklungs GmbH or prepared byreaction of chitosan with a suitable thiolated reactant, e.g., asdescribed in the above-mentioned Published PCT Application No. WO03/020771 A1 or in the above-mentioned Roldo et al., Krauland et al.,Bernkop-Schniirch and Bernkop-Schnürch et al. papers.

The chitosan desirably is obtained in dry particulate form, for example,as free-flowing granules whose average particle diameter is less thanabout 1 mm, less than about 100 μm, about 1 to about 80 μm, or less than1 μm. The chitosan preferably is packaged and shipped to a user in suchdry particulate form so as to reduce degradation of the chitosan duringprolonged storage. The chitosan fluid may be formed for example bydissolving the chitosan in water or another suitable solvent just priorto use. Recommended chitosan amounts will depend on the chitosanmolecular weight, and may for example be about 1 to about 20%, about 1to about 10% or about 1 to about 5% of the resulting solution. U.S.Published Application No. US 2009/0291911 A1 describes a preferredtechnique for rehydrating a chitosan, by dispersing free-flowingchitosan particles in a biocompatible water-miscible polar dispersant,and combining the dispersion with sufficient aqueous solvent for theparticles to convert them to a cohesive hydrogel. The chitosan may becomminuted but desirably is non-comminuted.

A wide variety of oxidized polysaccharides may be employed in thedisclosed fluid layer and method. Exemplary polysaccharides includeagars, alginates, carrageenans, celluloses, chitins, chitosan (thusenabling chitosan to be crosslinked using its oxidized counterpart),chondroitin sulfates, dextrans, galactomannans, glycogens, hyaluronicacids, starches and other biocompatible polysaccharides capable of beingoxidized. Oxidized polysaccharides such as oxidized cellulose, chitin,chitosan, chondroitin sulfate, dextran, glycogen, hyaluronic acid andstarch are preferred. The polysaccharide desirably is oxidized to anextent sufficient to provide aldehyde groups capable of promoting rapidcrosslinking of the chitosan when the chitosan and oxidizedpolysaccharide are combined in aqueous solution. Representativeoxidizing agents or techniques include the use of a) sodium periodate,b) hypochlorite ion in the presence of di-tert-alkylnitroxyl catalysts,c) metal-catalyzed oxidation, using for example ruthenium, d) anhydrousoxidation using for example nitrogen dioxide in for example ahalocarbon, e) enzymatic or chemo-enzymatic oxidation of starch, guarand other polysaccharides, and other oxidation agents and techniquesthat will be known to persons having ordinary skill in the art.Depending on the selected oxidizing agent or technique, a variety ofdegrees of oxidation, degrees of polymerization and oxidation sites maybe employed. For example, oxidation may be directed at a primaryhydroxyl group (for example, the 6-hydroxyl group in the anhydroglucoseunits of glucans), resulting in carboxyl-polysaccharides with preservedring structures. Oxidation may also be directed at a vicinal diolfunction present in a monosaccharide ring (for example, the C2-C3 sitein anhydroglucose units), resulting in cleavage of the monosaccharideunits and the production of dialdehyde or dicarboxyl functional groups.The dialdehyde content of such an oxidized polysaccharide may range froma degree of oxidation of, for example, 2% to virtually 100%, e.g., morethan 30% or more than 50% of the available oxidation sites. The oxidizedpolysaccharide may also contain other functional groups, for examplehydroxyalkyl groups, cationic groups, carboxyl groups and other acidgroups. As a generalization, reduced amounts of oxidized polysaccharidemay be employed in the disclosed fluid layer and method as the degree ofpolysaccharide oxidation is increased.

The oxidized polysaccharide desirably is dissolved in water or anothersuitable solvent prior to use. Recommended oxidized polysaccharideamounts typically will depend on the oxidized polysaccharide molecularweight, and may for example be about 1 to about 20%, about 1 to about10% or about 1 to about 5% of the resulting solution. The oxidizedpolysaccharide solution normally is kept separate from the chitosansolution until just prior to use.

Compared to crosslinking using a low molecular weight aldehyde such asglutaraldehyde or genipin, oxidized polysaccharides appear to providefaster gelation while avoiding the use of potentially less bioacceptablelow molecular weight aldehydes. In addition to their ability to reactwith amine groups in the chitosan, aldehyde groups in the oxidizedpolysaccharide may also enhance mucoadhesion. The oxidizedpolysaccharides may provide additional benefits including improved orbetter controlled biodegradability, bioresorbability, drug delivery orhaemostatic properties. The presence of phosphate ions appears toaccelerate the crosslinking reaction. Phosphate may be provided by usingphosphate buffered saline (PBS) as a solvent for one or both of thechitosan and oxidized polysaccharide.

Sufficient chitosan and oxidized polysaccharide desirably are employedso that a protective gel layer will form in less than 30 minutes afterthe chitosan and oxidized polysaccharide are mixed, and more preferablyin less than 20 minutes, less than 10 minutes, less than 5 minutes oressentially immediately after mixing. The resulting fluid mixture mayfor example contain chitosan and oxidized polysaccharide in a combinedamount representing about 1 to about 20%, about 1 to about 10% or about1 to about 5% of the composition. The chitosan and oxidizedpolysaccharide may for example be combined in a ratio of about 10:1 toabout 1:20, about 5:1 to about 1:10, or about 3:1 to about 1:5. Theseratios depend on the degree of oxidation of the oxidizedpolysaccharide(s), with lower oxidized polysaccharide amounts generallybeing used when more highly-oxidized polysaccharides are employed. Forsome applications the chitosan amount will preferably be as high as maybe feasible in order to provide good antimicrobial properties, and insuch cases it will be preferable to use a low amount of highly oxidizedpolysaccharide so as to obtain rapid gel formation.

The disclosed compositions desirably are substantially collagen-free.Preferably the compositions are sufficiently free of collagen (e.g.,containing no collagen at all) so as to be saleable worldwide for usewithout restriction in humans.

The disclosed compositions may optionally include a variety of otheringredients. These other ingredients may be disposed before mixing inthe first part, second part or both parts of a two-part composition.Exemplary other ingredients include water and other solvents (e.g.,alcohols), acids, bases, buffering agents, antimicrobial agents,therapeutic agents and other adjuvants. An acid, base or buffering agentmay for example maintain the composition at an appropriate pH forcontacting human tissue, e.g., a pH greater than 5, a near-neutral pH,or a pH less than 8.5. Exemplary buffering agents include barbitonesodium, glycinamide, glycine, potassium chloride, potassium phosphate,potassium hydrogen phthalate, sodium acetate, sodium citrate, sodiumphosphate and their conjugate acids.

The disclosed compositions desirably are inherently antimicrobialwithout requiring addition of a separate antimicrobial agent.Antimicrobial activity may be influenced by the proportion of chitosanin the composition (with higher chitosan proportions tending to providegreater antimicrobial activity) and by the number of available chitosanamine hydrogen atoms. Accordingly, use of chitosan derivativescontaining low numbers of available amino hydrogen atoms (such as theN-carboxyethyl derivatives desired in the above-mentioned Weng et al.paper) may be contraindicated. In any event, a separate antimicrobialagent may be employed if desired. A useful list of such antimicrobialagents may be found, for example, in the above-mentioned U.S. PatentApplication Publication No. US 2007/0264310 A1.

Exemplary therapeutic agents which may be employed in the disclosedcompositions include any material suitable for use at the intendedtreatment site including analgesics, anti-cholinergics, anti-fungalagents, antihistamines, steroidal or non-steroidal anti-inflammatoryagents, anti-parasitic agents, antiviral agents, biostatic compositions,chemotherapeutic/antineoplastic agents, cytokines, decongestants,hemostatic agents (e.g., thrombin), immunosuppressors, mucolytics,nucleic acids, peptides, proteins, steroids, vasoconstrictors, vitamins,mixtures thereof, and other therapeutic materials that will be known tothose skilled in the art. A useful list of such therapeutic agents maybe found, for example, in the above-mentioned U.S. Patent ApplicationPublication No. US 2007/0264310 A1.

Other adjuvants that may be included in the disclosed compositionsinclude dyes, pigments or other colorants (e.g., FD & C Red No. 3, FD &C Red No. 20, FD & C Yellow No. 6, FD & C Blue No. 2, D & C Green No. 5,D & C Orange No. 4, D & C Red No. 8, caramel, titanium dioxide, fruit orvegetable colorants such as beet powder or beta-carotene, turmeric,paprika and other materials that will be known to those skilled in theart); indicators; flavoring or sweetening agents including but notlimited to anise oil, cherry, cinnamon oil, citrus oil (e.g., lemon,lime or orange oil), cocoa, eucalyptus, herbal aromatics (e.g., cloveoil, sage oil or cassia oil), lactose, maltose, menthol, peppermint oil,saccharine, sodium cyclamate, spearmint oil, sorbitol, sucrose,vanillin, wintergreen oil, xylitol and mixtures thereof; antioxidants;antifoam agents; and rheology modifiers including thickeners andthixotropes. The disclosed compositions desirably do not containingredients which might potentially harm mucosal tissues or structures,e.g., tissues in the nasal or sinus cavities.

In those instances where it is desirable to remove water from tissue,e.g., to remove fluid from polyps or edematous tissue, a hyperosmolaragent may be employed in the disclosed compositions. Exemplaryhyperosmolar agents include furosemide, sodium chloride gel and othersalt preparations that draw water from tissue or substances whichdirectly or indirectly change the osmolar content of the mucous layer.Where sustained release or delayed release of a therapeutic agent isdesirable, a release agent modifier may also be included.

The disclosed composition typically will be subjected to sterilizationand placed in suitable sealed packaging (for example, a multicomponentsyringe, a vial or vials, or a multi-chamber pouch made of suitablematerials) prior to shipment to an end user. Additional propertycustomization may be carried out by using a sterilization procedure suchas gamma radiation or electron beam (E-Beam) processing to causecontrolled chain scission. Cold ionizing radiation sterilization (e.g.,cold E-Beam sterilization) may be employed to limit the degree of chainscission, as discussed in PCT Published Application No. WO 2009/132229A2. Whether or not sterilized, the first part containing the chitosannormally will be kept separate from the second part containing theoxidized polysaccharide until just prior to use.

The disclosed compositions may desirably be used as a part of amulti-step treatment regimen which disrupts a bacterial biofilm anddiscourages its return. For example, a series of steps that may bebroadly classified as Cleansing/Disrupting, Killing, Aerating,Protecting/Coating, and Healing may be carried out. TheCleansing/Disrupting step may be carried out by administering asolvating system as discussed above in connection with FIG. 1 and FIG.2. The Killing step may be carried out by applying a suitableantimicrobial agent to the treatment site. This may for example beaccomplished by including an antimicrobial agent in the solvatingsystem, as a separately-applied composition, or in both the solvatingsystem and in a separately-applied composition. An antimicrobial agentmay also be applied or administered post operatively. The Aerating stepmay be carried out by providing air passageways or improving airpassageways to the treated tissues by opening occluded or partiallyoccluded passages, e.g., the sinuses or sinus ostia for nasalapplications. This may for example be accomplished by surgicallyremoving obstructive tissue structures or by manually displacing suchstructures. The Protecting/Coating step may be carried out by coating atleast part of the thus-treated tissue with the disclosed compositioncontaining chitosan and oxidized polysaccharide as described above. TheHealing step may be carried out by allowing the cleansed, protected andsealed tissue surface to undergo a return to a normal state, e.g.,through one or more healing mechanisms such as modulation of aninflammatory response, phagocytosis, mucosal remodeling, reciliation orfull or partial restoration of normal function. The multi-step treatmentregimen may include or be followed by a Clearing step in which thedisclosed composition containing chitosan and oxidized polysaccharide issufficiently biodegradable or bioresorbable to disappear from thetreatment site in a desired time period, e.g., more than 1 day, morethan 3 days, or about 4 to 7 days, and desirably without shedding largesolid chunks. The disclosed method may advantageously be accomplishedwithout requiring surgery, for example by applying and removing theoptional solvating system and by applying the disclosed compositioncontaining chitosan and oxidized polysaccharide through normalaspiration/suction techniques or by simple flushing of affected tissue.A comparable series of steps may be performed in a multi-step treatmentregimen in a portion of the middle or inner ear. Further detailsregarding such a regimen may be found in U.S. Patent ApplicationPublication No. US 2007/0264310 A1.

The invention is further illustrated in the following non-limitingexamples.

Example 1 Gel Formulations

Chitosan solutions were prepared by dissolving varying amounts ofchitosan glutamate (PROTASAN™ UP G 113 or PROTASAN UP G 213 from theNovaMatrix unit of FMC BioPolymer AS) overnight in PBS. An oxidizedstarch (OXST) solution was prepared by dissolving P9265 polymericdialdehyde (from Sigma-Aldrich) in PBS while heating at 80° C. for 1-2hours. Oxidized methylcellulose (MC) and oxidizedhydroxypropylmethylcellulose (HPMC) solutions were prepared by reactingMC or HPMC with sodium periodate, lyophilizing the resulting products,and dissolving the lyophilized products in PBS. The resulting chitosansolutions and oxidized polysaccharide solutions were mixed in variousratios and concentrations. Rheological measurements determined thegelling time and storage modulus (G′) for the resulting hydrogels. Theresults are shown below in Table 1.

TABLE 1 Gelation Time and Storage Modulus G′ Chitosan: Oxidized OxidizedPoly- Total Gel Run Poly- saccharide Conc. time G′ No. Chitosansaccharide Ratio (%) (min) (Pa) 1 G 113 OXST 2:1 5 <2¹ 5,200 2 G 113OXST 1:1 5 <2¹ 4,000 3 G 113 OXST 1:2 5 <2¹ 4 G 113 OXST 2:1 3.75 <2¹800 5 G 113 OXST 1:1 3.33  3.2 400 6 G 113 OXST 1:2 3  6.4 200 7 G 213OXST 2:1 2.5 <2¹ 750 8 G 213 OXST 1:1 2.5 <2¹ 450 9 G 113 Oxidized MC1:2 7.5 <1 day 10 G 113 Oxidized MC  1:10 5 <2¹ 11 G 113 Oxidized MC 1:55 <2¹ 100 12 G 113 Oxidized MC 1:1 5 <1 day 13 G 113 Oxidized MC 5:1 5no gelling 14 G 113 Oxidized 1:4 12.5 <60 HPMC 15 G 113 Oxidized 1:2 7.5<1 day HPMC 16 G 113 Oxidized 1:1 5 no HPMC gelling ¹Gelled before therheometer measurement started

Each of the chitosan/oxidized polysaccharide formulations shown in Table1 had a viscosity below 500 cP, and should be injectable at a mucosaltissue treatment site using for example a device like that shown in FIG.2 or a variety of other devices that will be known to persons havingordinary skill in the art. The formulations shown in Table 1 should alsobe sprayable onto a mucosal treatment site. Due to the rapidity withwhich gelation occurred for some formulations, spray application wouldin many instances be a preferred mode of application. The Run No. 2formulation (G 113 Chitosan/OXST 1:1 at a total concentration of 5%) wasspray-applied using a gas-assisted applicator (FibriJet™ SA-6030regulator, from Micromedics, Inc., controlling a FibriJet SA-3652 sprayset equipped with a pair of 3 cc syringes). The OXST solution was dyedusing toluidine blue to make the applied fluid layer easier to see. Athin, rapidly formed strong gel was obtained.

Example 2 Antimicrobial Properties

The Run No. 7 and 8 formulations from Table 1 (G 213 Chitosan/OXST 2:1and 1:1 at a total concentration of 2.5%, respectively shown as bars Band C in FIG. 3) were evaluated to determine their antimicrobialactivity versus S. Aureus, using a plate procedure whose detection limitwas log 2. The gel formulations were placed in duplicate under sterileconditions directly into a 24-well polystyrene tissue culture plate.Each well was incubated with 1 mL (25,000 colony forming units) of abacterial suspension of S. Aureus (ATCC 25923). Positive controls wereincubated with 1 ml of trypticase soy broth (TSB). After 6 hoursincubation at 37° C., the media was transferred in new tubes and serialten-fold dilutions were performed. Ten μL aliquots from the appropriatedilution were plated in triplicate on trypticase soy agar plates usingthe dilution track method (Jett B. D. et al., Biotechniques, 23, 648-650(1997). The plates were incubated at 37° C. for 24 hours and ColonyForming Units (CFU) were counted. As shown in FIG. 3, both formulationsexhibited complete (greater than 6 log reduction) killing of thebacteria vs. the TSB control (see bar A in FIG. 3).

The Run Nos. 2, 10 and 11 formulations from Table 1 (G 113 Chitosan/OXST1:1, G 113 Chitosan/Oxidized MC 1:10 and G 113 Chitosan/Oxidized MC 1:5at a total concentration of 5%, respectively shown as curves B, C and Din FIG. 4) were evaluated as described above to determine theirantimicrobial activity as a function of time versus S. Aureus, using a140,000 CFU/mL bacterial loading, with measurements being recorded at 1,3 and 6 hours against a TSB control (see curve A in FIG. 4). As shown inFIG. 4, complete killing was observed after 6 hours for gels made fromall three chitosan/oxidized polysaccharide formulations, withsignificant killing being observed after 3 hours. The G 213Chitosan/OXST formulation appeared to provide faster killing than the G113 Chitosan/Oxidized MC formulations.

Example 3 Drug Delivery

The Run No. 1, 2 and 3 formulations from Table 1 (G 113 Chitosan/OXST2:1, 1:1 and 1:2 at a total concentration of 5%, respectively shown ascurves A, B and C in FIG. 5) were used to prepare drug-loaded hydrogelsin PBS buffer by mixing the chitosan and oxidized starch solutions withdexamethasone phosphate as the drug to be delivered. As shown in FIG. 5,relatively fast but controlled release for up to 3 days was obtained fordexamethasone phosphate, possibly aided by an interaction between theanionic drug and the cationic chitosan polymer.

Example 4 Degradation

The degradation behavior of various chitosan/oxidized polysaccharide gelformulations was determined by placing the gels in various buffersystems, including PBS at pH 7.4 with and without lysozyme (1 mg/mL),PBS with lipase, 2-(N-morpholino)ethanesulfonic acid (MES) at pH 6.0,and trishydroxymethylaminomethane (TRIS) at pH 7.4. Weight loss wasdetermined at various time points up to 28 days. Weight loss occurredusing all buffer systems, with about 30-60% of the original (dry) sampleweight remaining after 28 days. Gels with higher oxidized starch contentexhibited greater weight loss. The weight loss results forchitosan/oxidized polysaccharide in PBS at pH 7.4 and 37° C. (withoutlysozyme) are shown in FIG. 6.

The oxidized starch-based gels appeared to degrade into a ‘shell-like’material, whereas the oxidized cellulose-based gels appeared to remainas gels during degradation. The oxidized cellulose-based gels may behaemostatic.

The results in Examples 1-4 show that chitosan and oxidizedpolysaccharides may be combined to prepare injectable or sprayableformulations which quickly form strong protective gel layers in situwith inherent antimicrobial properties. The formulations were in eachinstance sprayable, antibacterial, biodegradable or bioresorbable andcapable of serving as a scaffold for drug delivery.

Although specific embodiments have been illustrated and described hereinfor purposes of description of the preferred embodiments, it will beappreciated by those of ordinary skill in the art that a wide variety ofalternate or equivalent implementations calculated to achieve the samepurposes may be substituted for the specific embodiments shown anddescribed without departing from the scope of the present invention.This application is intended to cover any adaptations or variations ofthe preferred embodiments discussed herein. Therefore, it is manifestlyintended that this invention be limited only by the claims and theequivalents thereof.

We claim:
 1. A method for treating bodily tissue or structure in theear, nose, sinus or throat, which method comprises: a) applying to suchear, nose, sinus or throat tissue or structure a fluid layer containinga mixture of (i) chitosan having a number average molecular weightbefore sterilization of at least about 50 kDa and (ii) an oxidizedpolysaccharide, and b) allowing the mixture to crosslink in situ to forma protective gel layer that remains exposed to air or other nearbygases, reduces adhesions to anatomy, and has a residence time of one dayto four weeks.
 2. A method according to claim 1 comprising applying thefluid layer to a nasal or sinus cavity.
 3. A method according to claim 1comprising applying the fluid layer to a middle or inner ear.
 4. Amethod according to claim 1 comprising applying the fluid layer tothroat tissue.
 5. A method according to claim 1 comprising applying thefluid layer to mucosal tissue.
 6. A method according to claim 1comprising applying the fluid layer from a multiple-barrel syringe.
 7. Amethod according to claim 1 comprising applying the fluid layer byspraying.
 8. A method according to claim 1 wherein the chitosan is anunmodified chitosan or a chitosan salt.
 9. A method according to claim 1wherein the chitosan is a chitosan derivative.
 10. A method according toclaim 1 wherein the oxidized polysaccharide comprises oxidized starch.11. A method according to claim 1 wherein the oxidized polysaccharidecomprises oxidized cellulose.
 12. A method according to claim 1 whereinthe oxidized polysaccharide comprises oxidized chitin, oxidizedchitosan, oxidized chondroitin sulfate, oxidized dextran, oxidizedglycogen or oxidized hyaluronic acid.
 13. A method according to claim 1wherein the chitosan and oxidized polysaccharide are combined in a ratioof about 10:1 to about 1:20.
 14. A method according to claim 1 whereinthe chitosan and oxidized polysaccharide are combined in a ratio ofabout 3:1 to about 1:5.
 15. A method according to claim 1 wherein thechitosan is a low molecular weight material having a number averagemolecular weight of about 50 to about 200 kDa.
 16. A method according toclaim 1 wherein the chitosan is a medium molecular weight materialhaving a number average molecular weight of about 200 to about 500 kDa.17. A method according to claim 1 wherein the chitosan is a highmolecular weight material having a number average molecular weightgreater than about 500 kDa.
 18. A method according to claim 1 whereinthe fluid layer remains a gel during biodegradation or bioresorbtion.19. A method according to claim 1 wherein the protective gel layer isbiodegradable or bioresorbable without shedding large solid chunks. 20.A method according to claim 1 wherein the fluid layer contains chitosanand oxidized polysaccharide in a combined amount of about 1 to about 10wt. % of the fluid layer.
 21. A method according to claim 1 wherein thefluid layer consists essentially of chitosan, oxidized polysaccharide, asolvent selected from water and alcohols, and an optional bufferingagent.